Figure 6. Bmal1 and Clock regulate anagen progression in hair follicle cycling.

(A) Representative histological sections of dorsal skin from Bmal1 ^−/− mice and their gender-matched Bmal1 ^+/− littermates at the indicated postnatal age (P). (B) Representative histological sections of dorsal skin from Clock mutant and their gender-matched wild-type (WT) littermates at the indicated postnatal age (P). For (A) and (B), time points are mapped (indicated by dotted line) based on histology to the corresponding phases of the hair growth cycle: anagen (A), catagen (C), and telogen (T). (C) Delayed anagen progression in Bmal1 ^−/− mice compared to normal progression in Bmal1 ^+/− littermates. At postnatal day 24, hair follicles are in anagen IIIb for the shown Bmal1 ^+/− dorsal skin section; matrix cells (black arrow) form the enlarged hair bulb and the dermal papilla (red arrow) is larger than a third of bulb diameter. Note that the bulb is located in the middle of the subcutaneous adipose layer. Hair follicles are in anagen I for the shown Bmal1 ^−/− dorsal skin section; thickening of keratinocyte strand (blue arrow) between the dermal papilla (red arrow) and the club hair. Note that the bulb is located in the dermis. Brackets indicate the different layers of the skin: E – epidermis, D – dermis, SC – subcutaneous adipose layer. (D) Quantitative hair cycle histomorphometric analysis. Percentage of hair follicles at the indicated hair cycle stage is based on staging fifty unique hair follicles for each genotype from three Bmal1 ^+/− and two Bmal1 ^−/− littermates at P24. Tel – telogen. Ana – anagen (Roman numerals indicate specific stages within anagen). (E) Q-PCR of Ccnd1, Ccnb1, and Myc in P24 Bmal1 ^−/− and Bmal1 ^+/− dorsal skin. Expression is normalized to Gapdh and error bars represent the S.E.M. for independent measurements from six Bmal1 ^+/− and two Bmal1 ^−/− littermates. Asterisks denote statistically significant (P<0.01) difference in expression between the two genotypes. Immunostaining of dorsal skin from P24 Bmal1 ^+/− (left panel), Bmal1 ^−/− (central panel) littermates using anti-phospho-histone H3 (F) and anti-phospho-Rb (Ser807/811) (G). The right panels of A and B are wild-type mice at P23 with hair follicles at equivalent stage of the hair growth cycle (anagen I) to the P24 Bmal1 ^−/− mice. The insets are higher magnification of the lower regions of hair follicles. Black arrowheads; cells stained positive in the epidermis. Black dashed line; border between epidermis and dermis. Red dashed line; hair follicle bulb. Red arrowhead; cells stained positive within the hair follicle.